This is a competitive renewal of NIH grant DK55545 which is focused on the role of PI 3-kinase in insulin action. PI 3-kinase is a critical node in insulin action in control of metabolism and a key point of divergence of insulin signaling. In previous work under this grant using both in vitro and in vivo approaches, including creation and characterization of mice and cell lines in which specific isoforms of PI 3-kinase regulatory and catalytic subunits have been deleted, we have demonstrated that this enzyme controls insulin signaling in both positive and negative ways, and is involved in much more than simply generation of PIP3. This includes differences in the activity and properties of the different regulatory (p85) and catalytic (p110) subunits of PI 3-kinase; important effects of stoichiometry between regulatory and catalytic subunits on insulin action; the ability of PI 3-kinase to allow divergence of the downstream signal between Akt and atypical PKCs; and alterations in PI 3-kinase activity in disease states. In addition, we have identified several previously unrecognized links between the PI 3-kinase pathway and other signaling pathways, including differences between the p110a and p110b catalytic subunits in support of downstream insulin actions and important links between the p85 regulatory subunits and several pathways involved in insulin resistance, including the stress kinases JNK and p38; the major PIP3 phosphatase in cells and tissues PTEN; and a novel link between PI 3-kinase, endoplasmic reticulum (ER) stress and the unfolded protein response. The latter occurs through interaction of p85a with the transcription factor XBP-1 and can modify the ER stress response involved in insulin resistance. This has led us to new hypotheses about the important role of the different PI 3-kinase catalytic and regulator subunits as both sites of divergence in the insulin signaling pathway and sites of both positive and negative regulation in physiological and pathological states, as well as sites for cross-talk with other signaling systems. In the next five years, we will expand upon these observations at both the molecular and physiological levels by defining how different signals are generated by different regulatory and catalytic subunits of PI 3- kinase, the specific signaling complexes involved, and the role of this system in vivo in insulin resistant and diabetic states. Specificall we will further elucidate the link between PI 3-kinase regulatory subunits and induction of ER stress by defining the specific pathways and molecules interacting with the p85 regulatory subunits in vivo and muscle and determining if p85 plays a role in the ER stress response in pancreatic b-cells in diabetes and other states with altered insulin secretion. This will be done i vitro and in vivo through creation of b-cell specific p85a KO mice crossed with a mouse that secretes a mutant insulin molecule or mice with states of obesity and hyperinsulinemia. We will also characterize the first human mutation in p85 associated with severe insulin resistance and determine the differential roles of the PI 3-kinase catalytic subunits p110a and p110b in divergent insulin signaling in the PI 3-kinase pathway.